Development of a High Sensitivity SPE-LC-MS/MS Assay for the Quantification of Glucagon in Human Plasma Using the ionKey/MS System

Introduction

Glucagon for Injection (rDNA origin) is a polypeptide hormone identical to human glucagon and is used to treat severe hypoglycemia (low blood sugar).¹ It is a single chain polypeptide that contains 29 amino acids residues with a molecular weight of 3483 (Figure 1). As a research tool, accurate quantification of glucagon from biological matrices can help us to better understand diabetes as a function of disease progression and/or drug treatment. Many assays, using different methodologies, exist for glucagon analysis in biological samples.2-7 Glucagon, like other biologics, has historically been quantified using ligand binding assays (LBAs).2-5 With advances in MS and chromatography technologies over the past few years there has been a trend toward the analysis of large molecules by LC-MS/MS. This is in part driven by the fact that LBAs can suffer from significant cross-reactivity issues and lack of standardization. Additionally, LC-MS/MS also has the advantage of shorter development times, greater accuracy and precision, the ability to multiplex, and can readily distinguish between closely related analogues, metabolites or endogenous interferences. Large peptides, such as glucagon, are particularly difficult to analyze by LC-MS/MS as MS sensitivity is low due to poor transfer into the gas phase and poor fragmentation. In addition, glucagon suffers from significant non-specific binding, poor solubility, and must be properly stabilized in biological matrices during collection and sample preparation,6-8 making LC and sample preparation method development challenging.